There is a compelling biomedical need for additional high resolution structural information on G protein-coupled receptors. Preliminary data is presented from collaboration between the labs of C. Sanders, T. Iverson, and R. Breyer showing that many human Class A G-protein coupled receptors (GPCRs) can be heterologously overexpressed and purified in quantities sufficient for structural biology, and that it is possible to obtain both crystals and solution NMR spectra of the receptors. However, it is clear from our preliminary data that novel tools and approaches are required to improve sample conditions so as to attain fully functional receptor and to enhance the quality of the structural data. The aims of this proposal are dedicated to developing the requisite novel technology. A premium is placed on developing methods that are inexpensive and easy to use, so as to maximize their accessibility by as many labs as possible. The new approaches are also designed to be modular, so that they can be readily integrated with each other and with the best of existing technology. We also focus on approaches that will be equally useful in X-ray crystallography, NMR spectroscopy, and other biophysical approaches to GPCR structure and mechanism. Specific Aim 1. Develop methods to replace the critical native disulfide bond found in most Class A GPCRs with alternative structural elements. This will allow functional receptor to be generated following exogenous expression without first having to achieve native disulfide pairing, which is often very difficult. Specific Aim 2. Test novel surfactants that are designed both to stabilize GPCRs under micellar and bicellar conditions, and to mimic functionally-essential components of native membranes. Bicelle-forming bipolar surfactants will be tested as a route to enhancing receptor stability. We will also test new micelle-compatible cholesterol derivatives for their ability to fulfill the functionally-essential role that cholesterol plays for many receptors under native conditions. Specific Aim 3. Develop methods for refolding GPCRs that are based on mimicking the iterative approach taken by nature to achieve folding of eukaryotic membrane proteins. In particular, we will develop artificial folding chaperones for GPCRs. These aims will be accompanied by solution NMR and crystallization screens, so as to directly evaluate structural biological outcomes as new methods are developed.